Malate thiokinase catalyzes ATP dependent thiol ester formation between Coenzyme A and a number of dicarboxylic acids; a reaction analogous to the succinate thiokinase reaction. The purified enzyme has been shown to be composed of equimolar amounts of two non-identical subunits, giving a subunit structure of alpha 4 beta 4 for the enzyme. Phosphorylation of the ezyme by ATP, occurs on the alpha subunits, as does phosphorylation of succinate thiokinase. Phosphorylated malate thiokinase, like phosphorylated succinate thiokinase, liberates phosphate in acid, but is stable in base, consistent with the formation of phosphohistine. Sucrose density gradient centrifugation studies show that phosphorylation of malate thiokinase leads to dissociation of the enzyme into a dimer whose subunit composition is alpha 2 beta 2. Reformation of the alpha 4 beta 4 tetramer can be accomplished by diphosphorylation of the enzyme with succinate, malate, or ADP. Non-covalent binding of coenzyme A or succinyl CoA also causes dissociation of the tetramer into the dimeric alpha 2 beta 2 form. Thus it appears that although native malate thiokinase is a tetramer whose structure is alpha 4 beta 4, the catalytically active form of the enzyme has the same subunit structure of succinate thiokinase from E. coli, namely an alpha 2 beta 2 structure.